This invention relates to methods for measuring the plasma volume of a human or animal and to compounds for use in such methods.
The adequate oxygenation of tissue depends on cardiac output, the concentration of haemoglobin and total circulating blood volume (BV). The concentration of haemoglobin is often expressed as the Haematocrit (Hct) or packed cell volume (PCV). At a given cardiac output, both haematocrit and total circulating blood volume are important determinants of tissue oxygenation. A low haematocrit means a low oxygen carrying capacity and a low blood volume leads to under perfusion of some tissue in order to maintain the oxygenation of others such as brain and cardiac muscle. The estimation of blood volume is therefore an important measurement in patients with threatened tissue oxygenation. Total blood volume (BV) is the sum of red cell volume (RCV) and plasma volume (PV) and they are related to the whole body haematocrit (Hct) by the following relationship:                     Hct        =                              RCV                          PV              +              RCV                                =                      RCV            BV                                              Equation (1)            
It is known to assess the plasma volume (and thus indirectly the blood volume) using the plasma protein, albumin. Albumin has been labelled with various dyes, including Evans Blue (T1824) and Indocyanin Green, as well as being radio-labeled with iodine. A known amount of the labelled albumin is injected into the bloodstream and then a dilution analysis technique used to determine the volume of distribution of albumin. However, we have noted that, especially in sick patients, equating the volume of distribution of albumin to plasma volume is unsatisfactory in that the albumin also equilibrates into Interstitial water and also xe2x80x9cleaksxe2x80x9d at a rate of about 5% per hour and increasing to 15% per hour in patients with septic shock. The albumin space exceeds true plasma volume and this is exaggerated when capillary integrity is impaired. The xe2x80x9cexcessxe2x80x9d volume of distribution of labelled albumin reflects mainly extravasation of the albumin simultaneously with its equilibration in the plasma in the circulation, in critically ill patients.
Accordingly, in present hospital practice worldwide, surgical resuscitation after blood loss is managed without accurate knowledge of measured blood volume. Likewise, intensive care patients who are dependant on respiratory support are managed without accurate knowledge of blood volume, but instead surrogate indicators of blood volume may be used such as clinical appraisal and intravascular pressure and flow measurements. These are only very generally related to the amount of blood in the circulation and it is possible for sick patients, including surgical cases or victims of trauma and the newborn, to have a deficiency of more than half the blood in their circulation.
Without any realistic possibility of routine measurement of plasma volume and thus total circulating blood volume, the effects of intravenous infusions used to attempt correction are hard to assess. Lack of this information seriously limits clinical monitoring and appraisal of the circulation of the most critically ill patients, as well as of less seriously sick patients who have undergone elective surgery.
The consequences of lack of knowledge of the circulating blood volume include management of patients who are hypo-volaemic with adverse effects on lung function and gastrointestinal tract, hepatic and renal perfusion by blood. In our own experience and observation, hypo-volaemia is extremely widespread in critically ill patients and goes undetected by present means of clinical evaluation and laboratory measurement.
Other methods of blood volume measurement are available which require the use of radioactive isotopes or non-radioactive xe2x80x9clabelsxe2x80x9d to determine the amount of red cells and plasma in the circulation. Such methods include measuring red cells by monitoring dilution of cells labelled with radioactive chromium or technetium, or with non-radioactive biotin. Plasma is routinely measured from dilution of radiolabelled albumin. All of these measurements are expensive in expertise, time and financial cost and they do not lend themselves to widescale application. Furthermore, most of the standard methods are unsuitable for use in women in the childbearing age group or in children.
Accordingly, there is a need for a method of blood volume assessment with a more widely applicable and speedy methodology, which is less prone to error in critically ill patients and which can be implemented at relatively low cost and for 24 hours every day. Whilst the traditional methods of blood volume measurement using radiolabelled albumin are reasonably accurate in healthy patients, as noted above we have found that the accuracy declines considerably in sick patients, who are usually in most need of accurate blood volume measurement.
We describe below methods of determining the plasma volume of a human or animal, which methods comprise introducing into the bloodstream of the human or animal, labelled material comprising a modified or unmodified starch, or derivatives or mixtures thereof, and thereafter determining the concentration of said labelled material, thereby to determine the plasma volume.
In the course of our experiments we have found that, in sick patients, the plasma volume determined by use of labelled albumin is higher than that using labelled starches of somewhat larger molecular size We believe that this is due to the increased capillary permeability in sick patients arising from damage to the vascular endothelium, with albumin extravasating into the interstitial space which accounts for about 25% of the water volume of a normal adult. This has severe implications not only for the accuracy of any measurements of plasma volume using labelled albumin, but also for the use of albumin as a volume expander in patients undergoing major surgical procedures. A rapid and profound increase in systemic capillary permeability is now known to accompany all acute inflammatory states including major surgery, ischaemia and reperfusion injury, trauma, thermal injury, bacteraemia, and acute pancreatitis.
The problem with albumin infusion as a volume therapy stems from the ease with which albumin moves from the vascular compartment to the interstitium, even in health, and the loss of vascular endothelial integrity in disease. Of the 275 grams of albumin in a normal adult, 60% is to be found in the interstitial space, with 5 grams or more every hour moving across the normal vascular endothelium. This rate of leakage dramatically increases in any severe inflammatory condition leading to hypoalbuminaemia and interstitial oedema. Intravenous infusions of albumin, given in an attempt to correct blood loss during an acute inflammatory episode such as trauma, major surgery or their complications, are not only ineffective because albumin rapidly leaks out of the vascular compartment, but also worsen interstitial oedema, with each gram of albumin xe2x80x98bindingxe2x80x99 18 grams of water. The blood in the underfilled circulation, depleted of plasma and red blood cells, gives misleadingly xe2x80x98normalxe2x80x99 haemoglobin and haematocrit values, suggesting that red blood cell replacement has been adequate even though may be a deficit of up to 20-30% and typically 1.5-2.5 litres in an adult patient. The interstitial oedema and the inadequate red cell mass compromise both pulmonary gas exchange and tissue oxygen delivery. Hypovolaemia leads to reflex vasoconstriction, and splanchnic ischaemia, risking bacterial translocation and ineffective function of the reticuloendothelial system.
This clinic xe2x80x98ITUxe2x80x99 (Intensive Therapy Unit) syndrome of xe2x80x98symmetricalxe2x80x99 red cell and plasma depletion with hypovolaemia, hypoalbuminaemia and interstitial oedema yet normal haematocrit and haemoglobin is clinically unrecognizable until its late stages which may herald organ failures. Conventional management of hypovolaemia advocating albumin infusions without blood transfusion does not provide a sustained increase in intravascular volume since, in the xe2x80x98sickxe2x80x99 ITU patient with damaged vascular endothelium, albumin rapidly moves out of the vascular compartment and exacerbates interstitial oedema, especially in the lungs, gastrointestinal tract wall, kidneys and skin. Recognition of the failure of the vascular endothelium and the reduced blood volume should highlight the need for transfusion of red blood cells together with colloids which remain in the vascular compartment even during periods of capillary leak.
Furthermore, recent work by Gosling et al (Gosling P., Bascom J. V., Zikria, B. A, Capillary Leak, Oedema and Organ Failure; breaking the triad; Care of the Critically Ill 1996: 191-197) shows that capillary leak is of prime importance in the development of systemic inflammatory response syndrome (SIRS) and multiple system organ failure (MSOF) and that its early detection should allow its correction through replacement of deficient red cells and plasma.
Accordingly there is a need for a method of determining the capillary leakage of a patient, which can be effected easily and rapidly.
International Patent Application WO 96/15721 describes a method for determining plasma volume in which labelled macromolecules stated to be larger than the endothelial junctions in the patient""s capillaries are introduced into the patient""s blood stream and a dilution analysis carried out. However it will be noted from FIG. 1 of the document that the concentration of the macromolecular material reduces to around 70% of the starting value which indicates that the method would lack accuracy and that a proportion of the molecules supposed to remain in circulation are escaping.
International Patent Application WO 96/16588 describes a method for determining the presence of capillary leak in which a tracer material having molecules smaller than epithelial gaps in he capillary of a patient is introduced into the patient""s blood stream and blood samples taken at intervals to ascertain the change in concentration of the tracer material with time over a time period from 30 minutes to 150 minutes. This method implies that capillary leakage can be determined by looking for differences in the rate of removal.
However our own data indicate that, over extended periods, the rate of removal of a polydisperse substance such a hydroxyethyl starch is independent of the molecular weight and of the presence or otherwise of capillary leakage, and therefore that this method would not be effective. In addition, the above disclosures apparently assume a one stage model in which the capillary vessels have gaps through which the smaller molecules may pass in an irreversible capillary leak.
The experiments below have led us to conclude that the mechanism is actually rather more complex, involving two different mechanisms. In a first stage molecules of smaller size are adsorbed into the capillary wall (for example by the smaller molecules binding to receptors or occupying pores). During this first stage (typically within the first 10 minutes), the concentration declines very quickly and the amount of the tracer material removed is dependent on the dose, the molecular size of the tracer materials and the presence of tissue oedema. We believe that the amount of tracer material removed in this way indicates the level of inflammation)capillary permeability, and is greater with small molecules and during endotoxaemia. This phenomenon is exemplified by an increase in the binding of small molecules to endothelial cell walls and/or a small increase in the number and/or size of the pores.
In the second stage, there are gross changes in pore size and density, and the molecules undergo a reversible equilibration between plasma and interstitial water, and the decline in concentration during the first hour or so represents this process.
The method of International Patent Application WO 96/16588 apparently assumes that the tracer macromolecules are either in circulation in the plasma or have leaked from the capillary into the interstitial space. However, this does not allow for molecules which become adsorbed into the capillary wall during the first stage, which we have found is an extremely important indicator of the onset of the stage two of the process, i.e. capillary leak syndrome. Thus tests such as that of International Patent Application WO 96/16588 do not indicate capillary leak until it has actually occurred, when it may be too late to save the patient.
Accordingly, methods which provide an early indicator of the onset of capillary leak during stage one could considerably improve patient treatment, by early transfusion of red blood cells as appropriate.
Thus the preferred embodiments below provide two estimates of the plasma volume; a first estimate using a low dose, low molecular weight tracer which gives an apparent volume of plasma distribution which varies according to the extent of adsorption of tracer molecules in receptor binding sites or small pores (and also leakage from the capillary), and a second estimate using a high dose high molecular weight tracer which gives a true plasma volume which is independent of surface adsorption and leakage effects. By comparing these two volumes a measure of capillary permeability is obtained which will flag the onset of capillary permeability indicated by increased surface adsorption etc., before the second stage occurs.
In one aspect, this invention provides a method of determining the presence or onset of capillary permeability of a subject which comprises:
introducing into the blood stream of the subject a dose of a first labelled macromolecular material;
taking at least one blood sample after said introduction;
determining the amount and/or concentration of said first labelled macromolecular material;
introducing into the bloodstream of the subject a dose of a second labelled macromolecular material having a relatively high molecular weight;
taking at least one blood sample after said introduction;
determining the amount and/or concentration of said second labelled high molecular weight macromolecular material;
using the relative determined concentrations and/or amounts of said labelled materials to indicate the presence or onset of capillary leakage.
Preferably, said low molecular weight tracer material and said high molecular weight tracer material comprise physiologically acceptable colloid or crystalloid materials, wherein, in humans or animals exhibiting capillary permeability, substantially all of said high molecular weight material remains within the capillary, whereas a substantial proportion of said low molecular weight material is capable of extravasation from plasma or adsorption on the capillary wall. Said low and high molecular weight materials may each comprise modified or unmodified polysaccharide. A particularly preferred material comprises hydroxyethyl starch which has already been cleared for clinical use as a plasma substitute or expander.
Hydroxyethyl starch has a branched and highly hydrated structure which makes it larger than other molecules of similar molecular mass. Preferably, said starch is polydisperse, with a weight-average molecular weight [MWw], expressed in Daltons (Da), between about 200,000 and 450,000 and a number-average molecular weight [MWn] between about 60,000 and 70,000 with MWw and MWn towards the lower ends of the ranges being particularly preferred. MWw is analogous to the mean of all molecular weights and is therefore influenced by the weight of large molecules. MWn is analogous to the median and is not influenced by a small proportion of very large molecules. A suitable material is 6% Elohaes(trademark) available from Fresenius (UK) Limited in Chester, UK.
Said low and high molecular weight materials advantageously comprise respective low and high molecular weight fractions of a polydisperse colloid or crystalloid material.
Preferably said low molecular weight tracer material contains a substantial proportion of molecules of molecular weight less than 100,000, and said high molecular tracer material contains a substantial proportion of molecules having a molecular weight greater than 100,000.
Although various marking or labelling techniques may be used, said low and high molecular weight tracer materials preferably each include a fluorescent label, for example fluorescein isothiocyanate. To allow use of the same or similar labels and to reduce the time between introducing said materials, without the first test masking the results of the second, said low molecular weight tracer material is preferably introduced at a relatively low concentration, and said high molecular weight tracer materials is introduced at a relatively high concentration after the first sample has been taken, thereby to distinguish between said low and high molecular weight tracer materials.
Alternatively the labels of said low and high molecular weight materials may be different and mutually distinguishable, whereby said low and high molecular weight tracer materials may be introduced together into the bloodstream.
The invention also extends to a kit for use in a method as described above, the kit comprising:
(i) a container containing a low molecular weight labelled tracer material having a relatively low molecular weight, and
(ii) a container containing a high molecular weight labelled tracer material having a relatively high molecular weight.
Said high molecular weight material preferably comprises a high molecular weight fraction of a polydisperse starch, said fraction containing a substantial proportion of molecules having a molecular weight of greater than 100,000, and said low molecular weight material comprises a low molecular weight fraction of said polydisperse starch, said fraction containing a substantial proportion of molecules with a molecular weight of less than 100,000.
Whilst the invention has been described above, it extends to any inventive combination of the features set out above or in the following examples.
The examples are given for illustration only and are not intended to limit the scope of the invention in any way,